Single molecule interaction between carbohydrate-binding modules and plant cell wall cellulose

Abstract

The world now greatly needs new sources of liquid transportation fuels to solve the economic and environmental problems. Enzymatic degradation of lignocellulosic biomass, mainly plant cell walls, has been considered as the most promising process for biofuel production. However, this process is greatly limited by the natural resistance of plant cell walls and inefficient enzyme-cellulose interactions. The carbohydrate-binding modules (CBMs) in the carbohydrate-active enzymes can facilitate the accessibility of enzymes by specifically binding to the target carbohydrates. As the beginning step in cellulose hydrolysis, the binding affinity and mechanism of this process is critical, but still unclear down to molecular level. Therefore, understanding single molecular CBM-cellulose interaction is greatly needed to improve the enzymatic hydrolysis of biomass. In this study, a specific and reliable single-molecule approach was applied to study the CBM-cellulose binding interactions. With a CBM-functionalized AFM tip, the binding specificity and affinities of two CBMs to poplar cell wall crystalline cellulose were determined by AFM recognition imaging and single molecule dynamic force spectroscopy (SMDFS). Several dynamic and kinetic parameters were quantified on natural and extracted plant cell wall cellulose at single-molecule level, such as unbinding forces, reconstructed free energy change, energy barrier, and bond lifetime. Specifically, a CBM3a molecule showed slightly higher binding efficiency and affinity than those of a CBM2a molecule to both natural and extracted crystalline cellulose. Both CBMs showed higher affinities to natural cellulose microfibrils than those to extracted cellulose microfibrils. The cell walls of poplar, switchgrass and corn stover before and after dilute acid pretreatment were also characterized. The results showed that the cell wall surface coverage of crystalline cellulose increased from 17-20% to 22-38% after pretreatment under different acid concentrations at 135 oC, and corn stover pretreated with 0.5% acid revealed an optimized distribution of crystalline cellulose on surface. A minimal effective CBM3a concentration of 5.1×10-7 M at a comparatively short reaction time of 287 min was also quantified for a more economic hydrolysis process. This study provides an in-depth understanding of the binding mechanism of CBMs to cellulose and may pave the way for advanced enzyme design and biomass degradation.